Characterization and expression of two cDNAs encoding carbonic anhydrase in Arabidopsis thaliana

Abstract

Two distinct cDNA clones encoding carbonic anhydrase (CA) were isolated from an Arabidopsis thaliana lambda YES library. One of these clones, CA1, encodes a 36.1-kD polypeptide and is essentially the same as a previously reported Arabidopsis CA cDNA (C.A. Raines, P.R. Horsnell, C. Holder, J.C. Lloyd [1992] Plant Mol Biol 20: 1143-1148). Comparison of the derived amino acid sequence from this clone with other plant CAs suggests the presence of a chloroplastic transit peptide, which, when cleaved, would render a mature protein of 24.3 kD. The other identified clone, CA2, encodes a 28.3-kD polypeptide, which in addition to other residue changes, is 78 amino acids shorter at the N terminus than the primary product of CA1. The two cDNAs exhibit 76.9% sequence similarity at the DNA level and 84.6% identity between the predicted amino acid sequences. A polyclonal antibody generated against pea CA (N. Majeau, J.R. Coleman [1991] Plant Physiol 100: 1077-1078) hybridized to two protein bands (25 and 28 kD) from a total leaf extract and to only one band (25 kD) from a chloroplastic protein extract. The data suggest that the CA2 protein is an extrachloroplastic form of CA, presumably localized in the cytoplasm. Southern analysis indicated that CA1 and CA2 are encoded by different genes. Northern analysis of total leaf RNA resulted in hybridization of CA1- and CA2-derived probes to two transcripts of 1.47 and 1.2 kb, respectively. These data provide additional evidence that the CA2 clone is a full-length cDNA and that two transcribed CA genes are present in the Arabidopsis genome. Transcript levels of CA1 and CA2 decreased 70 and 20%, respectively, when mature plants were transferred to dark for 24 h. Seedlings germinated in the dark showed CA1 and CA2 transcript abundance levels of 4 and 22%, respectively, when compared with light-germinated seedlings. These data suggest that expression of CA1 is light regulated and dependent of leaf and/or chloroplast development. A possible role for cytoplasmic CA in the plant cell is discussed.